The present invention relates to proteins that direct the secretion of virulence proteins of pathogenic bacteria such as Shigellae and the use of such proteins, peptides, and fragments thereof to detect, prevent and treat disease. The invention also relates to a method of determining the intracellular requirements of genes of pathogenic organisms that invade, colonize, persist and cause disease in the host.
Bacteria of the genus Shigella are gram negative enteric pathogens which are the causative agents of bacillary dysentery or shigellosis. Shigella infection accounts for a considerable fraction of acute diarrheal diseases worldwide and is an important public health problem in developing countries where bacillary dysentery remains a major cause of childhood mortality. The worldwide incidence of bacillary dysentery is estimated to exceed 200 million cases annually. About 5 million cases require hospitalization and about 650,000 persons die of shigellosis each year (Institute of Medicine, 1986). Shigellosis continues to be an important public health concern even in the United States with over 32,000 cases reported in 1995 (Centers for Disease Control, 1995). Of principal importance are food borne outbreaks and outbreaks in institutional settings (day care centers, nursing homes, ect.) and on Indian reservations. The clinical presentation of shigellosis can range from a mild diarrhea to severe dysentery with frequent passage of bloody, mucoid, small volume stools. The disease is characterized by extensive damage to the colonic epithelial layer, cell death, ulceration and inflammation of the colon. While infections are usually self-limit and do not spread from the lamina propria to the submucosa, shigellosis can be life-threatening in young or malnourished patients (DuPont et al., 1995). There exists no effective vaccine against shigellosis.
The primary means of human to human transmission of Shigella is by the fecal-oral route. Most cases of shigellosis are caused by the ingestion of fecally-contaminated food or water. In the case of foods, the major factor for contamination is the poor personal hygiene of food handlers, particularly in view of the low infectious dose of Shigella spp. Volunteer studies showed that the ID50 (the infectious dose required to cause disease in 50% of the volunteers) of Shigella is as low as 200 shigellae, although it has been reported that the ingestion of as few as 10 organisms is sufficient to cause disease (DuPont et al., 1989).
The low ID50 of Shigella accounts for its high communicability, particularly in impoverished and crowded populations. One consequence of this feature is that a contaminated food source has the potential to cause explosive outbreaks of dysentery with secondary cases likely to occur among close contacts of infected individuals. Thus, infected food handlers can contaminate food and spread infection among large numbers of individuals. Several examples of food borne outbreaks of shigellosis are described in Maurelli et al., 1997. In particular, day care workers and children attending day care facilities are placed at risk when a child infected with Shigella is present. The bacteria are shed in feces and the immature personal hygiene habits of very young children can easily lead to infection of other children as well as care providers (Mohle-Boetani et al., 1995).
With a low infectious dose required to cause disease coupled with oral transmission via fecally-contaminated food and water, it is not surprising that dysentery caused by Shigella spp. follows in the wake of many natural (earthquakes, floods, famine) and man-made disasters (war). Civil wars in Burundi and Rwanda led to massive movement of refugees. An outbreak of dysentery in a refugee camp in Rwanda in late 1993 affected more than 6,000 people (attack rate  greater than 32%), mostly children under five years old (Paquet et al., 1995). In August, 1994, more than 15,500 cases of bloody diarrhea were reported from three refugee camps in Zaire (Centers for Disease Control, 1996).
When natural or man-made disasters destroy the sanitary waste treatment and water purification infrastructure, developed countries assume the conditions of developing countries. These conditions place a population at risk for diarrheal diseases such as cholera and dysentery. Recent examples include the war in Bosnia-Herzegovina, and famine and political upheaval in Somalia (Levine et al., 1994). All of these factors are exacerbated by the fact that Shigellae are becoming increasingly resistant to most antimicrobial agents commonly used in the treatment of diarrheal diseases (Centers for Disease Control, 1994).
There are four species of the genus Shigella serologically grouped (39 serotypes) based on their somatic O-antigens: Shigella dysenteriae (group A; 10 O groups), S. flexneri (group B; 13 O groups), S. boydii (group C; 15 O groups), and S. sonnei (group D; 1 O type). As members of the family Enterobacteriaceae, they are nearly genetically identical to Escherichia coli and closely related to Salmonella and Citrobacter (Ochman et al., 1983). One class of E. coli, the enteroinvasive E. coli (EIEC), has pathogenic properties that are similar to Shigella. EIEC cause a disease that is clinically similar to bacillary dysentery, and these bacteria harbor a large plasmid that has the same genetic determinants for virulence as Shigella. EIEC share certain biochemical properties with Shigella such as being nonmotile and unable to synthesize lysine decarboxylase. In addition, some serogroups of EIEC share identical O-antigens with certain Shigella serotypes (Sansonetti et al., 1985). However, despite these differences, strains of EIEC and Shigella express many of the same biochemical characteristics as E. coli. This biochemical similarity can pose problems in distinguishing these pathogens from E. coli found in normal flora.
The clinical symptoms of shigellosis can be directly attributed to the hallmarks of Shigella virulence: the ability to invade epithelial cells of the intestine, multiply intracellularly, and spread from cell to cell. All of the genes required for the invasion step are encoded on a large virulence-associated plasmid that is present in virulent strains of all species of Shigella as well as EIEC. These plasmids are functionally interchangeable with respect to expression of the invasion phenotype and share significant degrees of DNA homology (Sansonetti et al., 1985). Studies have focused on the 220 kb virulence plasmid of S. flexneri 2a. A 37 kb region of the invasion plasmid has been found to contain all of the genes necessary to permit the bacteria to penetrate into tissue culture cells. This DNA segment was identified as the minimal region of virulence plasmid necessary to allow a plasmid-cured derivative of S. flexneri (and E. coli K-12) to invade tissue culture cells (Maurelli et al., 1985). The nucleotide sequence of this part of the virulence plasmid from S. flexneri, as well as one from S sonnei, is known (see Galan et al., 1995 for summary; GenBank accession #D50601 for S. sonnei). The region encodes about 33 genes contained in two groups of genes transcribed in opposite orientation (FIG. 1a).
The 37 kb region of the virulence plasmid includes genes for invasion plasmid antigens (ipaBCDA) that encode the immunodominant antigens detected with sera from convalescent patients and experimentally challenged monkeys (Oaks et al., 1986). ipaB, ipaC and ipaD have been experimentally demonstrated to be required for invasion of mammalian cells (Menard et al, 1993). ipaA is also necessary and although an ipaA mutant shows about a 10-fold decrease in its ability to invade HeLa cells, internalization is not completely impaired in an ipaA mutant (Tran Van Nhieu, et al. 1997). The Ipa products are found associated with the outer membrane of Shigella and in culture supernatants. IpaB and IpaC form a complex on the bacterial cell surface and trigger a eukaryotic membrane ruffling process responsible for mediating entry via bacterium-directed phagocytosis (Menard et al., 1994; Adam et al., 1995; Menard et al., 1996; Parsot et al., 1995). Unlike other invasive pathogens, like Salmonella spp., Shigella spp. lyse the post-phagocytic endosomal membrane and multiply in the eukaryotic cell cytosol (Sansonetti et al., 1986). It is in the cytosol where Shigella develops the ability to spread intercellularly. The bacterium uses a protein polarly localized in the outer membrane, the IcsA protein, to direct the polymerization of host cell actin monomers that serve as a motor, propelling the bacterium within cellular protrusions, or fireworks, into adjacent uninfected cells (Bernardini et al., 1989 and Goldberg et al., 1993). Protrusion escape, which requires the lysis of two cellular membranes (that of the primary and secondarily infected cells), establishes the infection in neighboring cells and leads to bacterial spread across the colonic mucosa (Allaoui et al., 1992a).
To function in invasion and spread, the products of the ipa genes are secreted into the extracellular medium. Secretion occurs despite the lack of signal sequences for recognition found in the usual gram-negative bacterial transport system. Indeed, a growing number of animal and plant pathogens have been found to be similar to Shigella in the production of outer membrane or secreted virulence proteins which lack classical signal sequences. In spite of this diversity, the mechanisms utilized for bacterial virulence protein delivery are remarkably homologous. Research has shown that in Shigella Ipa protein secretion occurs via a dedicated transport apparatus composed of the gene products from another locus on the virulence-associated plasmid (FIG. 1a). The membrane expression of invasion plasmid antigens/surface presentation of Ipa antigens locus, called the mxilspa locus of Shigella, encodes 20 Mxi and Spa proteins, many of which have been demonstrated to be essential to Shigella virulence as measured in the Sereny test, invasion of and proliferation in cultured epithelial cell lines, plaque assay, or the binding of Congo red dye. Mxi/Spa proteins comprise the machinery necessary for secretion of the Ipa products (Andrews et al., 1991; Allaoui et al., 1992; Allaoui et al., 1993; Sasakawa et al., 1993) and define a unique system for protein secretion in pathogenic gram negative bacteria which is designated the type III secretion system.
The type III secretion systems are membrane bound, multicomponent structures responsible for the translocation of virulence effectors from the bacterial cytoplasm to either cell surface or intracellular eukaryotic targets (Hueck, 1998). Type III secretion is often referred to as contact-dependent, as it is induced by direct pathogen-host interaction. Additionally, expression of such systems can be induced at the transcriptional level by certain host conditions, including temperature and salt concentrations. Loci encoding varying groups of homologous type III secretory apparatus components have been identified, usually within large operons, in many mammalian and plant pathogens, including Shigella, Salmonella, Yersinia, enterohemorrhagic and enteropathogenic Escherichia coli, Pseudomonas, Burkholderia, Chlamydia, Bordetella, Xanthomonas, Ralstonia, and Erwinia. Several well conserved components of type III secretion systems are also similar to proteins involved in flagella biosynthesis, a finding which supports the theory that type III pathways arose from those responsible for flagellar subunit secretion. The loss of virulence in mxi-spa mutant strains of Shigella is generally attributed to failure of the type III secretion apparatus to secrete Ipa proteins.
Despite this growing body of knowledge, there is still a need in the art for the identity and function of the proteins involved in the entry and spread of pathogenic bacteria like Shigellae. There is also a need for systems to determine the intracellular expression requirements of the genes that encode proteins involved with entry and spread of such bacteria to facilitate the development of regimens for the diagnosis, prevention, and treatment of disease.
The present invention relates to our discovery that the mxiM protein of Shigella flexneri is indispensable for host cell entry and the spread of Shigella from cell to cell. Thus, the invention provides the mxiM protein or peptides or portions thereof as antigens in vaccines to prevent Shigella infections and treat hosts infected with Shigella by inhibiting intercellular spread. In another aspect, the invention relates to antibodies generated against the mxiM proteins, peptides, or portions thereof to detect Shigella in contaminated food and water supplies as well as in infected hosts. In yet another aspect, the invention relates to the DNA sequence of the mxiM gene or a fragment thereof to detect Shigella in contaminated food and water supplies as well as in infected hosts.
The present invention also describes a method called the TIER (test of intracellular expression requirements) for determining the intracellular expression requirements of genes and therefore, permitting one to establish the role of genes in the pathogenesis of organisms.